Actin and myosin are highly conserved proteins found in virtually all eukaryotic cells where they generally exist as labile polymeric structures assembled into the cytoskeleton. They participate directly in determination of cell shape.cellular motility.cytoplasmic streaming.cytokinesis and contractility in muscle cells. The primary role of the interaction of actin and myosin is the generation of force and motion. This occurs as a consequence of the cyclic interaction of the motor domain of myosin with actin filaments and is driven by myosin ATP hydrolysis. Our research has concentrated on the analysis of myosin structure. assembly and function using protein biochemical. immunochemical and ultrastructural techniques. These efforts have lead to the determination of the high resolution structure of the myosin domain responsible for force generation and motion as a result of our work on the structure of myosin S1 crystals. We intend to continue our efforts along these lines by analyzing the conformational transitions within the myosin head which give rise to movement of actin filaments. This will be accomplished using monoclonal antibodies as site specific probes of the interaction of myosin with actin using an in vitro motility assay. The orientation of the myosin head when tightly bound to actin will be examined using cryo-electron microscopy to map the location of specific myosin sequences on rigor complexes of acto-S1 that are labeled with Fab fragments of monoclonal antibodies. These studies will provide landmarks for relating the crystal structure of myosin S1 to the structure of actin. The folding and assembly of the myosin molecule will be analyzed with an in vitro synthesis and assembly assay. These experiments are aimed at defining the role in the folding and stability of the molecule of highly conserved sequences in the myosin head and are derived from the observation that a mutation at one of these sites in the myosin S1 domain is related to a human genetic disease. Familial Hypertrophic Cardiomyopathy (FHC). To analyze the structural and functional consequences of mutations in the S1 domain. We will produce recombinant myosin fragments in eukaryotic expression system. Finally, the design and interpretation of these studies are based on the structure of myosin S1. We will continue our successful collaboration with Dr. Ivan Rayment on the analysis and refinement of the structure of the myosin head with the aim of defining the molecular mechanism used by myosin to generate force and motion.